Diagnosis of chronic obstructive pulmonary disease

ABSTRACT

Methods of determining Chronic Obstructive Pulmonary Disease (COPD) severity in patients which comprises measuring the concentration of soluble E-cadherin (sE-cadherin) in the patient&#39;s urine and/or blood serum and determining the extent of severity by reference to correlation graphs, a method of treating patients with COPD, methods for determining the responsiveness of said patients to said treatment and a prognostic product for detecting the concentration of sE-cadherin in urine and/or blood serum.

This invention relates to a novel method for prognosis of a patient witha respiratory disease, specifically chronic obstructive pulmonarydisease.

Chronic obstructive pulmonary disease (COPD) is a disease characterisedby chronic inflammation and irreversible airflow obstruction with adecline in the lung function parameter FEV1 that is more rapid thannormal. The disease has two major aspects of pathology, namely chronicbronchitis, characterised by mucus hypersecretion from the conductingairways, and emphysema, characterised by destructive changes in thealveoli.

Currently a number of pharmaceutical substances are indicated for orhave been shown to be useful in treating the symptoms of COPD, includingsalmeterol xinafoate, fluticasone propionate and ipratropium bromide.(2R,3R,4S,5R)-2-[6Amino-2-(1S-hydroxymethyl-2-phenyl-ethylamino)-purin-9yl]5-(2-ethyl-2H-tetrazol-5yl)-tetrahydro-furan-3,4-diolis also of development interest in the treatment of COPD, as aretiotropium,4hydroxy-7-[2-[[2-[[3-(2-phenylethyoxy)propyl]sulfonyl]ethyl]amino]ethyl2(3H)-benzothiazoloneand cis-204cyano4-[3-(cyclopentyloxy)4methoxyphenyl]cyclohexanecarboxylic acid.However there is considerable interest in evaluating the extent, if atall, these medicines are disease modifying i.e. affect the overallprogression of the disease either in terms of symptom severity orexacerbation severity.

Additionally many of the symptoms of COPD are shared by otherrespiratory diseases such as asthma, bronchitis, pulmonary fibrosis andtuberculosis. Accordingly COPD is considered to be a poorly diagnoseddisease and due to this fact a great number of patients are deniedmedicine that could be of benefit to them. In addition, there is a needfor new medicines that will be more effective than current medicines. Inview of the economic impact of COPD there is considerable incentive fordrug discovery in this area.

The presenting symptoms for COPD are breathlessness accompanied by adecline in FEV1. Chronic bronchitis can be diagnosed by asking thepatient whether they have a “productive cough” i.e. one that yieldssputum. Patients are traditionally treated with bronchodilators orsteroids and examined by spirometry for reversibility of airflowobstruction. If reversibility is less than 15%, and particularly if theyhave a long history of smoking, then they would be classified as COPDpatients.

The ATS (American Thoracic Society) criteria for diagnosing COPD are asfollows:

-   FEV1/FVC ratio<0.7-   FEV1<70% predicted, <15% reversibility to inhaled B2 agonist    PLUS:-   2 week oral prednisolone trial—less than 15% reversibility in FEV1-   Smoking history-   Excluding alpha-1 AT deficiency (by blood test)-   Non-atopic (skin tests) and no history of atopy-   Stable: without exacerbation for at least 6 weeks-   No history of childhood asthma

There is a need in the art to identify a reliable and straightforwardindicator of the COPD disease state (for example, a surrogate marker)both in order to reliably distinguish the symptoms of COPD from those ofthe above mentioned respiratory diseases and to predict changes indisease severity and progression, and response to medicine, before thesechanges are manifest clinically.

Elevated levels of cytokeratin 19 fragments have been detected in thebroncheoalveolar lavage fluid of patients with chronic inflammatory lungdisease and this observation was suggested as a marker of bronchialepithelial injury (Nakamura, H. et al., 1997: Am. J. Resp. Crnt. CareMed. 155, 1217-1221). However, no attempt was made to correlate levelsof this marker with lung function (e.g. FEV1).

The inventors of the present invention have surprisingly identified ahitherto unappreciated correlation between the concentration of solubleE-cadherin in blood serum and urine in a patient and the severity ofCOPD as measured by a reduction in the patient's FEV1.

FEV1 is the volume of air expelled from the lungs in one second,starting from a position of maximum inspiration and with the subjectmaking maximum effort. FEV1% is the FEV1 expressed as a percentage ofthe forced vital capacity (FVC). The FVC is the total volume of airexpelled from the lungs from a position of maximum inspiration with thesubject making maximum effort.

FEV1 may be measured using a spirometer to measure the volume of airexpired in the first second of exhalation.

E-cadherin is a member of the calcium dependent adhesion moleculesuperfamily and is expressed in epithelia, including those of the lung,gut and skin. It has a major role in controlling epithelialintercellular adhesion since it influences the formation of allepithelial intercellular junctions. Adhesion is mediated by interactionbetween extracellular domains of E-cadherin dimers on adjacent cells. Inthe adherens junction, cadherin dimers assemble in a zipper-like mannerincreasing the adhesive strength. In certain epithelialhyperproliferative conditions, there is some shedding of E-cadherinextracellular domains as soluble fragments, (sE-cadherin). Theconcentration of sE-cadherin in the circulation has been shown to beincreased in patients with certain turmours and also to correlate withthe PASI score (measure of disease severity) of psoriasis patients(Matsuyoshi, N. et al. (1995) Brit. J. Dermatol. 132, 745-749).

Concentration of E-cadherin in the blood serum or urine may bedetermined using a specific ELISAl Using this assay, the inventors haveshown a direct and inverse linear correlation between actual FEV1 inCOPD patients (as a percentage of the predicted value of FEV1) andsE-cadherin levels in serum and urine respectively.

The results of a trial demonstrating these correlations are described inExample 1 and shown in FIGS. 1 and 2.

Thus the concentration of soluble E-cadherin in blood serum and urine isa molecular indicator for COPD which is capable of reporting itsseverity without recourse to evaluating any symptom except reduction ina patient's FEV1.

The predicted (normal) FEV1 of a patient may be calculated by themethods determined by Morris JF et al 1971: Am Rev Resp Dis 103, 57-67based on given height and age. The values are influenced by age, sex andheight.

A patient already diagnosed as having COPD can be assayed for diseaseseverity at a time point by comparison of his concentration of solubleE-cadherin in blood serum or urine at that time point with the indicatorof severity shown in FIGS. 1 and 2.

Progression of COPD disease may be evaluated by monitoring theconcentration of soluble E-cadherin in blood serum or urine with time.

It will be appreciated that either the concentration of solubleE-cadherin in blood serum or urine may be measured for the prognosis,however the recordal of both measurements will be confirmatory of theprognosis. The strength of the confirmation is emphasised by the inversecorrelation between the two measurements as shown in FIGS. 1 and 2.

It will be appreciated that a particular and unique benefit of theinvention is the ease of prognosis which may be performed requiring onlya simple blood or urine sample.

Thus, according to the invention, we provide a method of determining theseverity of COPD in a patient which comprises measuring theconcentration of soluble E-cadherin in a sample of the patient's urineand determining the extent of severity by reference to a correlationgraph such as one which correlates FEV1 (as a percentage of thepredicted value) with soluble E-cadherin concentration eg. as shown inFIG. 2.

We also provide a method of determining the severity of COPD in apatient which comprises measuring the concentration of solubleE-cadherin in a sample of the patient's blood serum and determining theextent of severity by reference to a correlation graph such as one whichcorrelates FEV1 (as a percentage of the predicted value) with solubleE-cadherin concentration eg. as shown in FIG. 1.

For greater confidence, the method may comprise measuring theconcentration of soluble E-cadherin in a sample of the patient's bloodserum and urine and determining the extent of severity by reference to acorrelation graph for each such as one which correlates FEV1 (as apercentage of the predicted value) with soluble E-cadherin concentrationeg. as shown in FIGS. 1 and 2.

As a further aspect of the present invention we provide a method oftreating a patient suffering from COPD which comprises determining theextent of the disease by identifying the levels of soluble E-cadherin ina sample of the patient's blood serum followed by administration of acompound which ameliorates the symptoms of the disease.

We also provide a method of treating a patient suffering from COPD whichcomprises determining the extent of the disease by identifying thelevels of soluble E-cadherin in a sample of the patients urine followedby administration of a compound which ameliorates the symptoms of thedisease.

We also provide a method of treating a patient suffering from COPD whichcomprises determining the extent of the disease by identifying thelevels of soluble E-cadherin in a sample of the patient's blood serumand urine followed by administration of a compound which ameliorates thesymptoms of the disease.

As a further aspect of the invention we provide a method of determiningthe responsiveness of a patient with COPD to therapy which comprisesmonitoring the concentration of soluble E-cadherin in samples of thepatient's blood serum with time and determining the rate of change ofextent of progression of the disease by reference to a correlation graphsuch as one which correlates FEV1 (as a percentage of the predictedvalue) with soluble E-cadherin concentration eg. as shown in FIG. 1.

We also provide a method of determining the responsiveness of a patientwith COPD to therapy which comprises monitoring the concentration ofsoluble E-cadherin in samples of the patient's urine with time anddetermining the rate of change of extent of progression of the diseaseby reference to a correlation graph such as one which correlates FEV1(as a percentage of the predicted value) with soluble E-cadherinconcentration eg. as shown in FIG. 2.

For greater confidence, the method may comprise monitoring theconcentration of soluble E-cadherin in samples of the patient's urineand blood serum with time and determining the rate of change of extentof progression of the disease by reference to a correlation graph foreach such as one which correlates FEV1 (as a percentage of the predictedvalue) with soluble E-cadherin concentration eg. as shown in FIGS. 1 and2.

As a further aspect of the invention we provide a product for prognosisof COPD severity in a patient which comprises means to report theconcentration of soluble E-cadherin in a sample of blood serum takenfrom the patient.

We also provide a product for the prognosis of COPD severity in apatient which comprises means to report the concentration of solubleE-cadherin in a sample of urine taken from the patient.

We also provide use of means to report the concentration of solubleE-cadherin in a sample of a patient's urine in the manufacture of aprognostic product for determination of COPD disease severity in apatient.

We also provide use of means to report the concentration of solubleE-cadherin in a sample of a patient's blood serum in the manufacture ofa prognostic product for determination of COPD disease severity in apatient.

For blood serum analysis, a 20-30 μl volume of blood taken from a‘pin-prick’ would be suitable and for urine analysis a sample ofapproximately 1 ml taken “mid-flow” would be suitable.

Means to report the concentration of soluble E-cadherin in a sample ofblood serum or urine preferably comprises an anti-soluble E-cadherinantibody.

For example, sE-cadherin concentration may be measured using acommercially available kit from Takara. This kit allows the measurementof sE-cadherin, using standard ELISA technology and the standard curveprovided, which allows interpretation of the measurement in terms of aconcentration.

EXAMPLE 1

Blood serum, urine and induced sputum from 4 patient groups (healthynon-smokers, healthy smokers, asthmatics and COPD patients) were sampledand the soluble E-cadherin concentration in each body fluid wasmeasured.

FEV1 was measured using the method given above. Predicted (normal) FEV1was calculated for each patient in accordance with the algorithm givenin the above mentioned Morris et al (1971) paper and the actual FEV1given as a percentage of predicted.

Table 1 contains information relating to all patients used in thisexample.

Pack years refers to the level of smoke exposure. One pack year equatesto 20 cigarettes smoked per day for 1 year. The medicaments used in thetable refer to ‘salb’: salbutamol and ‘atro’: Atrovent (ipratropiumbromide).

The results are shown in the following Figures:

FIG. 1—FEV1 (as a percentage of the predicted value) as a function ofconcentration of soluble E-cadherin in blood serum

FIG. 2—FEV1 (as a percentage of the predicted value) as a function ofconcentration of soluble E-cadherin in urine.

The predicted value of FEV1 was determined according to Morris JF et al1971: Am Rev Resp Dis 103, 57-67.

The results presented in FIG. 1 show that FEV1 (as a percentage of thepredicted value) (y) is correlated with concentration of solubleE-cadherin in blood serum (x) in COPD patients according to Spearman'srank correlation analysis.

The correlation coefficient and p-values for the 4 patient groups fromthese data are as follows:

Corr coeff p-value Healthy non-smokers −0.36 0.521 Healthy smokers −0.230.307 Asthmatics 0.02 0.946 COPD patients 0.67 0.033

The results presented in FIG. 2 show that FEV1 (as a percentage of thepredicted value) (y) is correlated with concentration of solubleE-adherin in urine (x) in COPD patients according to Speannan's rankcorrelation analysis.

The correlation coefficient and p-values for the 4 patient groups fromthese data are as follows:

Corr. coeff. p-value COPD −0.66 0.038 Healthy Smokers −0.76 0.016Healthy Non-Smokers −0.57 0.088 Asthma −0.11 0.761

Both FIGS. 1 and 2 show that there is no correlation between FEV1 andconcentration of soluble E-cadherin in urine or blood serum inasthmatics.

TABLE 1 Mean [sE- Mean Mean cadherin] in [sE- [sE- sputum cadherin]cadherin] Mean [Urine]/ Medication FEV1 supernatant in serum in urineMean [Creatinine] [Creatinine] Age of Sex of taken by Pack % PatientGroup (ng/ml) (ng/ml) (ng/ml) [IL-8] (mmol/L) ratio patient patientpatient Years pred COPD 1 294 5082 1795 5049 8.1 222 59 F nil 35 60 COPD2 94 4728 1518 10661 1.3 1168 66 M salb, atro 44 66.2 COPD 3 810 62391900 3218 4.7 404 48 M nil 30 64.5 COPD 4 71 7297 255 3598 0.6 425 45 Mnil 30 75 COPD 5 830 5342 3066 548 17.5 175 47 M atro 25 (ex) 41 COPD 61749 6781 3603 4809 12.4 291 43 M nil 30 69 COPD 7 179 5208 996 153973.2 311 45 F nil 30 67.1 COPD 8 821 1761 5617 14056 10.9 515 54 M nil 4023 COPD 9 815 5198 2340 11147 2.8 836 56 F nil 40 40.4 COPD 10 658 27365034 2954 12.3 409 65 M salb, atro 30 (ex) 54 Mean values of 632 50372612 7144 7.4 476 53 7 M   — 35 56 Patient Group Healthy Smokers 1 5065612 1775 827 9.4 189 42 F nil 15 93 Healthy Smokers 2 547 5740 4568 22115 304 42 F nil 22 92 Healthy Smokers 3 376 4830 4310 796 17.5 246 38 Fnil 10 98 Healthy Smokers 4 463 5590 2609 716 4.4 593 35 F nil 15 100Healthy Smokers 5 446 5103 — 544 — — 48 F nil 20 90 Healthy Smokers 6837 4733 878 461 16 55 26 F nil 10 101 Healthy Smokers 7 1671 2761 7803362 3.5 223 24 F nil 11 105 Healthy Smokers 8 697 4856 1956 673 7 27933 F nil 15 98 Healthy Smokers 9 368 5585 1684 393 12.8 132 28 M nil 10109 Healthy Smokers 10 403 3431 9558 425 12.7 753 38 F nil 20 93 Meanvalues of 516 4824 3270 562 11.4 312 35 1 M   — 14.8 98 Patient GroupHealthy Non- 1 673 5951 842 704 7.4 114 28 F nil nil 101 Smokers HealthyNon- 2 1006 7463 3976 179 15 265 41 F nil nil 96 Smokers Healthy Non- 3654 2617 750 547 3 250 28 F nil nil 99.4 Smokers Healthy Non- 4 538 46972737 603 8 342 31 F nil nil 97 Smokers Healthy Non- 5 1118 6804 4022 20617.3 232 33 F nil nil 83 Smokers Healthy Non- 6 367 3544 3329 337 11.2297 21 F nil nil 104 Smokers Healthy Non- 7 782 8697 4639 667 12.4 37452 F nil nil 100 Smokers Healthy Non- 8 673 7357 4918 347 11.5 428 47 Fnil nil 95 Smokers Healthy Non- 9 976 4041 4963 683 8.5 584 43 F nil nil84 Smokers Healthy Non- 10 1375 6094 2199 584 20 110 28 F nil nil 99.4Smokers Mean values of 816 5726 3237 486 11.4 300 35 0 M   — — 95.9Patient Group Asthma 1 334 5041 4900 753 13.7 358 39 M salb nil 101Asthma 2 508 2027 3464 48 23 151 30 M salb nil 98 Asthma 3 240 6153 181773 7.1 256 22 F salb nil 89 Asthma 4 348 4168 1190 37 4.1 290 41 F salbnil 95 Asthma 5 577 5403 2228 528 10.3 216 27 M salb nil 94 Asthma 6 5844625 4003 539 11 364 27 M salb nil 92 Asthma 7 320 5101 3406 285 16.7204 33 F salb nil 84 Asthma 8 324 6190 320 496 — — 40 F salb nil 98Asthma 9 1767 4044 8039 2070 6.4 1256 37 M salb nil 89 Asthma 10 6936038 2034 125 8.5 239 21 F salb Nil 98 Mean values of 436 4879 3140 32011.2 370 32 5 M   — — 94 Patient Group

1. A method of treating a patient suffering from Chronic ObstructivePulmonary Disease (COPD) which comprises determining the extent of thedisease by identifying the levels of soluble E-cadherin (sE-cadherin) ina sample of the patient's blood serum or urine and determining theextent of severity by reference to a correlation graph which correlatesForced Expiratory Volume in the first second of expiration (FEV1) withsE-cadherin concentration followed by administration of a compound whichameliorates the symptoms of the disease.
 2. A method according to claim1, wherein said identifying step comprises identifying the concentrationof sE-cadherin in a sample of the patient's blood serum.
 3. A methodaccording to claim 1, wherein said identifying step comprisesidentifying the concentration of sE-cadherin in a sample of thepatient's urine.
 4. A method according to claim 1, wherein saididentifying step comprises identifying the levels of sE-cadherin in asample of the patient's blood serum and urine.
 5. A method according toclaim 2 wherein the correlation graph correlates FEV1 (as a percentageof the predicted value) with sE-cadherin concentration.
 6. A methodaccording to claim 3 wherein the correlation graph correlates FEV1 (as apercentage of the predicted value) with sE-cadherin concentration.
 7. Amethod according to claim 4 wherein the correlation graph correlatesFEV1 (as a percentage of the predicted value) with sE-cadherinconcentration.